LARGE PLASMIDS

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Dec 10 08:18:00 EST 1992


In article <1992Dec9.094125.24828 at wehi.edu.au>, lyons at wehi.edu.au writes...
>I have had difficulty growing large (>10 kb) plasmids.  Others seem to be
>able to get constructs of this size up.  Is this due to sequences 
>differences, or are there tricks associated with growing or isolating
>large plasmids.
> 
>Can anyone direct me bacterial strains that might be superior for this
>purpose, or to plasmid backbones that might do it for me.  
> 
>What about growth conditions?  Is amplification likely to help? 
>How about growing them cooler and slower?
> 
>Help!!
> 
>Thanks
> 
>Ian Lyons
>The Walter and Eliza Hall Institute
>Melbourne

Ian,
        Your posting it is difficult to answer because it lacks details,
strains used, media used, etc.  We constently grow large chimeric plasmids
and cosmids using various media, from "super-broth" to 2xTY to Luria Broth.
There was a recent article in one of the BRL Focus publications this past
year that discussed plasmid yield vs plasmid size in various media and
we have reproduced these expts.  For example, if you have a 3 kb plasmid
you typically get 3-time more plasmid than you would get with a 9 kb plasmid
under identical growth conditions.  This is a rough estimate but rather
consistent.
        A 10 kb plasmid should give a reasonable yield of a couple of
hundred micrograms range per liter of cells.  

A few other things to be aware of are:
        1. larger plasmid/cosmid constructs have a greater tendency to
                shear during the isolation so take care with trying to
                eliminate any "violent" shaking, vortexing, etc during
                the isolation.
        2. be sure you are using a RecA minus host as larger plasmids and
                cosmids sometimes WILL rearrange or even delete regions of
		the insert in RecA plus hosts.
	3. sometimes the antibiotic used to select for hosts containing
		plasmid with marker antibiotic resistance goes off, so
		be sure to use rather fresh antibiotic (Amp, Tet, etc)
		and use it at the Maniatas recommended concentration or
		even higher.

As for the plasmid of choice, pBR or pUC family vectors seem to work fine
as long as the host is RecA-.  Amplification with chloramph. or other
appropriate antibiotic somewhat improves the yield depending on the vector,
host and insert.  We've not tried different growth temperatures but
airation (violent vs gentle shaking) seems to have an affect depending
on the host.

Hope this gives some help.
Cheers........bruce
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